Formulations of aminoglycoside and fosfomycin combinations and methods and systems for treatment of ventilator associated pneumonia (vap) and ventilator associated tracheal (vat) bronchitis

ABSTRACT

The present invention is antibiotic compositions, ventilator-based systems and methods relating to ventilator-associated pneumonia (VAP) and ventilator-associated tracheal (VAT) bronchitis. Antibiotic combinations of fosfomycin and an aminoglycoside, preferably amikacin, are administered via an inline nebulizer within the airway of the ventilator. Humidified conditions create an improved aerosol mist to treat VAP and VAT.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/480,514which is a continuation-in-part of U.S. application Ser. No. 13/844,244filed Mar. 15, 2013, now U.S. Pat. No. 8,826,904, which is acontinuation-in-part of U.S. application Ser. No. 13/548,115 filed Jul.12, 2012, now U.S. Pat. No. 8,603,439, which claims the benefit of U.S.Provisional Application No. 61/572,225 filed Jul. 12, 2011. U.S. Ser.No. 13/844,244 is a continuation-in-part of PCT/US12/46559 filed Jul.12, 2012 which claims the benefit of U.S. Provisional Application No.61/572,225 filed Jul. 12, 2011, which applications are incorporatedherein by reference.

BACKGROUND

Considerable medical literature and clinical experience establish thatventilator associated pneumonia (VAP) is a feared and often fatalcomplication of mechanical ventilation. In the United States, over250,000 patients per year are stricken with VAP or approximately 800cases per million population. In Melbourne, the incidence in 2006 wasreported as 6.2 cases per 1,000 ventilator days, similar to the rate inthe United States. Sogaard O S et al., a binational cohort study ofventilator-associated pneumonia in Denmark and Australia, Scand J InfectDis (2006), 38:256-264). The mortality of VAP averages 25%. Therefore,in patients with a poor prognosis, a VAP diagnosis is a life-threateningcomplication.

The onset and rapid progression to VAP usually occurs after 3-5 days ofmechanical ventilation and starts with initial colonization of theairway with pathogenic bacteria. The initial colonization is followed bya purulent tracheobronchitis (also known as ventilator-associatedtracheobronchitis (VAT)) which rapidly progresses to VAP. VAT isconsidered a precursor to VAP and is characterized as tracheobronchitiswithout new infiltrates on the chest radiograph (Nseir, Nosocomialtracheobronchitis Current Opinion in Infectious Diseases 2009,22:148-153). However, not all VAT progresses to VAP, and not all VAP hada VAT precursor. In addition, pneumonia in a patient on a ventilatorthat was acquired in the hospital and or a nursing care facility priorto intubation and start of mechanical ventilation is often from the samehighly pathogenic bacteria seen in VAP. Our use of the VAP term includesthese patients as they have a similar course and prognosis of patientsthat develop pneumonia after initiation of mechanical ventilation.

In addition to patient mortality, VAP also prolongs ICU stays and istreated with high doses of intravenous antibiotics. However, the levelsof antibiotics that can be achieved in the respiratory tract withintravenous administration are severely limited and are often lower thanthe effective concentrations needed to treat VAP. Moreover, thecontinuing emergence of drug-resistant organisms, particularly inhospital settings, makes treatment with intravenous antibioticsincreasingly less effective. Specifically, the emergence ofmultidrug-resistant bacteria such as methicillin-resistantStaphylococcus aureus (MRSA), and highly virulent Gram-negativepathogens is increasing the morbidity of VAP.

Over the past twenty years, multiple investigator-sponsored trials haveattempted to study aerosolized antibiotics to either treat or preventVAP. (See Palmer et al. in Critical Care Medicine 2008, 36(7):2008-2013;Wood et al. in Pharmacotherapy 2002, 22(8):972-982; and Lu et al. inAJRCCM (Volume 184:106-115, 2011).) Meta analysis of these trials showsbenefit in decreasing ventilators days and improving other outcomes.Recently, Palmer et al. supra performed a randomized blindedplacebo-controlled trial to determine the impact of aerosolizedantibiotics on outcomes in patients with VAT and/or VAP. Forty-threepatients were randomized to receive aerosolized antibiotics or placebofor 14 days. Choice of the aerosolized antibiotics was based on Gramstain of the endotracheal aspirate. Vancomycin or gentamicin were usedin patients with Gram-positive and Gram-negative microorganisms,respectively. Both antibiotics were used if both Gram-positive andGram-negative microorganisms were present. Most of the 43 patients werealso treated intravenously with systemic antibiotics. The authors foundaerosolized antibiotics to be associated with significantly lower ratesof VAP at the end of treatment, reduced usage of systemic antibiotics,and earlier weaning of patients from the ventilator—leading to shorterstays in the ICU.

Palmer et al. also showed the advantage of a cocktail of antibiotics,specifically gentamicin and vancomycin, that have Gram-negative andGram-positive respective activity in treatment of VAP and VAT, as manypatients are infected with both Gram-negative and Gram-positivebacteria. Interestingly, lower rates of antimicrobial resistance werealso found in patients treated with aerosolized antibiotics, likely assuboptimal levels, commonly seen with intravenous administration, areknown to promote the development of bacterial resistance.

The delivery system used by Palmer et al. was a small particle size jetnebulizer—no longer manufactured—that introduced an additional 6 L/mairflow into the airway. Such a nebulizer is incompatible with manymodern ventilators because modern ventilators have sophisticatedcontrol, monitor, and feedback systems that carefully adjust airflow andpressures in the airway. A recent study by Lu et al. comparedceftazidime and amikacin, aerosol (n=23) vs. IV (n=17) in a small Phase2 trial in established Gram-negative bacteria and VAP. After 8 days ofantibiotic administration, aerosol and intravenous groups were similarin terms of successful treatment (70% vs 55%), treatment failure (15% vs30%), and superinfection by other microorganisms (15% vs 15%).Antibiotic resistance was observed exclusively in the intravenous group.The authors concluded that aerosol antibiotics have similar efficacy tointravenous (IV) delivery and likely lead to lower rates of bacterialresistance.

The poor results of aerosol adjunctive therapy or primary antibiotictreatment in VAP is not surprising, because intravenous antibioticspenetrate poorly into the sputum. Aerosol antibiotics generally have a100-fold higher sputum concentration than the maximum dose IV deliveryand usually with one-tenth the systemic exposure. Aerosolizedantibiotics are rapidly cleared from the respiratory tract, and theiruse can provide either very high concentrations in the lungs, which isdesirable to control bacteria, or very low concentrations. This avoidslong periods of sub-MIC antibiotic concentrations that lead to thedevelopment of resistance. However, to date, no aerosolized antibioticsfor VAP or VAT have been approved by regulatory authorities.

A promising combination of Gram-negative and Gram-positive antibioticsfor VAT and VAP would be the combination of an aminoglycoside andfosfomycin. (Baker U.S. Pat. No. 7,943,118 and MacCleod J AntimicrobialChemotherapy 2009, 64:829-836.) In patients with cystic fibrosis (CF)and Pseudomonas aeruginosa (a Gram-negative bacteria) infections, an 80mg fosfomycin/20 mg tobramycin dose delivered twice daily as an aerosolby a vibrating plate nebulizer (PARI® eFlow®) was effective indecreasing the bacterial burden of P. aeruginosa, and Staphylococusaureus over a 28-day treatment period (Trapnell et al., AJRCCM185:171-178, 2012). Other aminoglycosides may also be synergistic withfosfomycin; Cai (J of Antimicrobial Chemotherapy 64 (2009) 563-566)reported that in both an in vitro and a systemically treated ratpseudomonas infection model, fosfomycin potentiated the efficacy ofamikacin to an even greater extent than tobramycin.

In spontaneously breathing patients, the importance of a well-toleratedaerosol is also known. While mild cough can be tolerated in a patient ona ventilator, coughing increases the airway pressures, putting thepatient at risk for barotrauma. It is well known that hyperosmolarsolutions for nebulization can cause cough. In fact, a 7% hypertonicsaline solution having an osmolality of 2411 Osm/kg is used to inducecough to obtain sputum specimens or to promote airway clearance inpatients spontaneously breathing with lung disease. Lower osmolalitysolutions still cause cough. A formulation of fosfomycin/tobramycin withan osmolality of approximately 832 osm/kg when tested in CF patientscaused noticeable coughing in 10 of 41 patients, while a placebo ofnormal saline (Osm/kg of 310) produced coughing in only 3 of 40patients. Wheezing, a more several measure of bronchospasm, occurred in5 of 41 patients compared to none in the placebo group. (AMJ Respir CritCar Med 185:171-178, 2012.)

Therefore, although some combinations of antibiotics, includingfosfomycin and aminoglycosides, have been used, combinations for VAP andVAT have not been approved, and several problems remain to be solved.First, ventilator circuits almost invariably include a humidifier tohumidify the dry gas using sterile water coming from high pressure gassupplies prior to the gas entering the patient's airway. Humidificationof the air leads to hygroscopic growth of the aerosol particles. Manyparticles grow to a large size and “rain out” in the endotracheal andventilator tubing or, if delivered to the patient, deposit in the largeairways rather than lungs. See Miller et al., Am J Respir Crit Care Med168:1205-09 (2003). An endotracheal tube's internal diameter averages7-8 mm, much smaller than the diameter of a typical trachea. The smallerdiameter increases “rain out” of large >5 micron particles such thatthose aerosol particles never reach the patient. The efficiency penaltyof leaving the humidification circuit on with use of jet nebulizer withan average particle size of approximately 5 microns (at the nebulizerprior to growth due to humidification) has been estimated as a loss of50% of the aerosol. (Palmer et al. in Critical Care Medicine1998:26:31-38.) To avoid this problem, the obvious resolution is to turnoff the humidifier during aerosol antibiotic therapy, as was done in thetreatment studies noted above (Palmer, Wood, Lu, Miller, supra). This isa successful approach but carries the finite risk of a health careworker neglecting to turn humidification back on. Accordingly,hospitals, critical care facilities, and regulatory agencies wouldlikely require specific warning devices to maintain humidification,because non-humidified gas causes drying of secretions, which makesrecovery from pneumonia even more difficult. A method that allowscontinuous humidification is optimal and would increase patient safetyand treatment efficacy.

Second, improved airway tolerance is needed for patients with VAT andVAP. In the recent study of Lu et al., patients commonly breathe out ofsequence from the ventilator, likely caused by irritation from thetherapeutic aerosol. Eschenbacher (Eschenbacher et al., Am Rev RespirDis 1984, 129: 211-215) published that mild asthmatic patients willcough when exposed to an aerosol without a permeant anion (such aschloride) concentration greater than 20 meq/liter even if the aerosol isisotonic. Lu utilized sterile water to reconstitute the powderedantibiotics and did not use any saline that would provide a permeantanion in his formulation. Lu's approach was to heavily sedate thepatient, which is not optimal.

Third, Eschenbacher (supra) tested a hypertonic saline solution of 1232mOsm/liter in mild asthmatic patients and showed that cough andbronchospasm were common. Pretreatment with bronchodilators couldprevent bronchospasm but could not prevent cough. As noted above,coughing in the ventilator is not desirable, as it can cause high airwaypressures leading to pneumothorax or interfere with delivery of adequateventilation. The osmolality of a fosfomycin/tobramycin combinationcurrently being used in clinical studies of outpatient cystic fibrosis(CF) patients is approximately 832 mOsm/liter, far above the physiologicairway osmolality of approximately 310 mOsm/liter. The high osmolalityis due to the low MW of fosfomcyin, coupled with the formulation offosfomycin as a disodium salt. The disodium salt of fosfomycin has asolubility of 50 mg/mL of water; other salts including calcium areavailable, but have less solubility such that concentrated formulationsare not practical. The high osmolality formulation is used so that eachdose can be delivered in a 2 mL solution in a vibrating mesh nebulizerso treatment time for a CF outpatient is approximately 5 minutes. Moredilute solutions would require longer administration times, which leadto poor compliance and potentially less efficacy. Higher osmoticconcentrations have larger hygroscopic growth. Thus, if suchformulations were used in a ventilator, a large amount of >5 micronparticles would rain out in the tubing, and the remaining amount thatwas delivered would likely be irritating to the airways. A commonadverse event of cough was reported in the phase 2 CF study. In fact thehigh dose (160 mg fosfomycin/40 mg tobramycin, same osmolality in a 4 mLsolution) was very poorly tolerated in the CF study in spite of all thepatients pretreated with a bronchodilator to prevent bronchospasm.

Fourth, the epidemiology of resistance and goals of therapy are verydifferent in VAP and VAT compared to outpatient CF. Moreover, in thehospital setting, once bacterial resistance occurs, the spread ofresistant bacteria between patients is rampant and epidemics are common.Furthermore, the risk of aminoglycoside-related toxicity from thecumulative long-term dose is serious because CF patients are treatedchronically for years with tobramycin aerosols. See Baker et al., U.S.Pat. No. 7,943,118. In contrast, in VAT and in VAP, a patient is likelyto receive only a single, two-week course of antibiotics. Therefore,when used to treat VAP or VAT, limiting the dose of an aminoglycoside ina combination product, and relying on fosfomycin to increase bacterialkilling, risks the loss of efficacy of both drugs if a patient hasbacteria that are fosfomycin resistant. For example, in contrast withhigh ratios of fosfomycin to tobramycin disclosed in Baker, et al., anoptimal formulation in VAP and VAT would have enough of anaminoglycoside dose to be an independently effective antibioticcombination. However, this approach would only increase the osmolalityof the formulation if the ratio of the formulation is held constant. Theadvantage of the fosfomycin would be to further enhance Gram negativekilling, including biofilms (Cai, supra) and to also treat Gram positivebacteria, including methicillin resistant Staphylococcus aureus (MRSA).Another advantage of fosfomycin is that it reverses some of the sputumantagonism that limits the bioavailability of aminoglycosides (MacCleod,supra), (Mendelman Am Rev Respir Dis 1985, 132:761-5). Thus, even if thebacteria is fosfomycin resistant, there may be some clinical benefit tothe combination by increasing the bioactive concentrations of theaminoglycoside.

One solution would be to dilute the formulations and increase the volumeplaced in the nebulizer for treatment. However, observation of a patientduring therapy is likely to be standard protocol. ICU specialist nursesor respiratory therapists would likely be required to observe thepatient during treatment adding additional costs to the therapy due tothe prolonged administration time. Serious adverse events can occurduring aerosol therapy, such as in Lu's study, where a patient had acardiopulmonary arrest due to a clogged exhalation filter on theventilator. An optimal formulation would have shorter delivery time thanthat of a dilute formula. Triggering the delivery during inspirationwould extend treatment time but the time loss may be offset byimprovement in the efficiency of delivery. Thus, a need for a treatmentprotocol exists wherein lower doses may be evaluated.

Accordingly, a need exists for antibiotic compositions, equipment, andtreatment methods and systems to alleviate or prevent VAT and VAPdespite the known challenges and the recognized risks.

SUMMARY OF THE INVENTION

The present invention is an improved formulation of an aminoglycoside,and specifically improved amikacin with fosfomycin in combinations,systems, and methods for the treatment, alleviation, and prevention ofventilator associated pneumonia (VAP) and ventilator associated tracheal(VAT) bronchitis. The antibiotic compositions of the invention includecombinations of specifically formulated amikacin and fosfomycin,combined in a hypertonic solution having specific concentrations andratios, predetermined concentrations of permeant ion designed to betolerant upon inhalation, including specifically chloride ion(CI-concentrating), pH ranges, particle sizes in an aerosol mist, andosmolality levels designed to further the therapeutic goals of theinvention. These physical and chemical parameters are uniquely selectedto enhance the bacteria static and bactericidal performance of the drugcombinations in both ventilator-based and nebulizer-based modes ofadministration. Specifically, the ratios of amikacin to fosfomycin aregreater than 1:1, greater than 9:5, greater than 2:1, and preferablygreater than or equal to 2.5-2.6:1.0. The pH range is generally betweenabout 4.4 and 7.5 and preferably between 6.9 and 7.4. The concentrationof permeant and ion is greater than 30 equivalents per liter and, insome formulations, greater than 40 milliequivalents per liter. Theosmolality is greater than 300-310 mOsm/Liter and less than about 800mOsm/Liter and generally less than 1,000 mOsm/Liter. The concentrationof the first and second antibiotic component is both individually andsynergistically in combination, bactericidal, and preferably having aquantity greater than MIC 90 for a target organism. The aerosol can beformed from a solution containing any low molecular weight drug thatrequires high concentrations for efficacy, or cations or anions of suchdrugs having an osmolality that is higher than desired for toleranceupon aerosol administration. In certain embodiments described below, theantibiotic components may be either liquids, solids, or formulated asaerosols or dry powders and may be any physiologically compatible saltof the compositions described herein.

The first component of the antibiotic combination and composition isamikacin, a well-known and widely used aminoglycoside having activityagainst Gram-negative organisms. Although amikacin is not approved foraerosol use, it has been used in multiple VAP studies as regimencomponent that includes either standard IV drugs or ceftazidime aerosol.See Niederman, et al., NKTR-061 (Inhaled Amikacin) Reduces IntravenousAntibiotic Use in Intubated Mechanically Ventilated Patients DuringTreatment of Gram-Negative Pneumonia. from 27th International Symposiumon Intensive Care and Emergency Medicine Brussels, Belgium. 27-30 Mar.2007 Critical Care 2007, 11 (Suppl 2):P97, 5, Lu Q, et al., Nebulizedceftazidime and amikacin in ventilator-associated pneumonia caused byPseudomonas aeruginosa in AJRCCM Articles in Press. Published on Apr. 7,2011 as doi:10.1164/rccm.201011-1894OC). Systemic exposure is low withaerosolized amikacin and thus safer than intravenous administration inregards to renal toxicity. A data base of greater than 15,000 hospitalpathogens was recently published and represents current resistance dataafter a generation of amikacin use Zhanel G G, et al., Antimicrobialsusceptibility of 15,644 pathogens from Canadian Hospitals: results ofthe CANWARD 2007-2009 study. Diagnostic Microbiology and InfectiousDisease 69 (2011) 291-306). The MIC 90 (The minimal inhibitoryconcentration of 90% of the isolates) was 32 μg/ml for Pseudomonas. Inall other Gram negatives with the exception of Stenotrophomonasmaltophilia, the MIC 90 was lower. The MIC 90 of S. maltophilia was >64μg/ml. The limitations of amikacin are that its activity against MRSA islimited, and activity against Gram negative bacteria in biofilms ispoor. Also, amikacin formulated for intravenous use is the sulphate ofthe amikacin base and is not ideal for inhalation therapy becausesulphate is not a permeant anion. Accordingly, the amikacin base isformulated with chloride as the counter anion for tolerability andefficacy. See Examples 6 and 7 below.

The second antibiotic component of the drug formulation is fosfomycin, abroad spectrum phosphonic acid antibiotic that has both Gram-positiveand Gram negative activity. Fosfomycin oral monotherapy is commonly usedto treat uncomplicated urinary tract infections. Recently fosfomycin wasproven to be safe and effective as an aerosol in combination withtobramycin in treating CF patients with pseudomonas infections. TrapnellB C et al., Fosfomycin/Tobramycin for Inhalation (FTI): Efficacy Resultsof a Phase 2 placebo-controlled Trial in Patients with Cystic Fibrosisand Pseudomonas aeruginosa. Poster 233 24th Annual North American CysticFibrosis Conference, Oct. 21-23, 2010, Baltimore, Md., Trapnell B C etal., Fosfomycin/Tobramycin for Inhalation (FTI): Safety Results of aPhase 2 placebo controlled Trial in Patients with Cystic Fibrosis andPseudomonas aeruginosa. Poster 234 24th Annual North American CysticFibrosis Conference, Oct. 21-23, 2010, Baltimore, Md. In addition,fosfomycin was effective against MRSA coinfection in approximatelyone-third of treated patients. The antibiotic's efficacy with amikacinis superior to what is seen with tobramycin. Cai et al. reported thatfosfomycin in vitro increased the activity in vitro of amikacin by afactor if 64, and in a rat biofilm pseudomonas infection model thecombination of fosfomycin and amikacin improved efficacy compared tomonotherapy of either component. Cai Y et al. Synergistic effects ofaminoglycosides and fosfomycin on Pseudomonas aeruginosa in vitro andbiofilm infections in a rat model. J of Antimicrobial Chemotherapy 64(2009) 563-566. Fosfomycin is not used in North America as an IVantibiotic, and there is no recent data on fosfomycin MICs from MRSA.However, data from the 1980s reports an MIC 90 of 32 μg/ml. Alvarez S etal., In Vitro activity of Fosfomycin, Alone and in Combination, againstMethicillin-Resistant Staphylococcus aureus. Antimicrobial Agents andChemotherapy 28 (1985) 689-690). With little general use, one wouldexpect similar values today, i.e., not showing development ofsubstantial resistance.

In one embodiment, the combined formulation will be a neutral pHhypertonic solution of at least about 50 mg/mL of amikacin with chlorideas the counter anion, and at least about 20 mg/mL of fosfomycin with atleast 30 equil/L of chloride anion. The osmolality of this formulationwill be approximately 700 mOsm/L, and with dilution from thehumidification from the ventilator circuit, the final osmolality will beapproximately 425 mOsm/L. Normal airway osmolality is 310 mOsm/L, andmildly hypertonic solutions are well tolerated by patients. The use of apermeant anion is to prevent cough in patients with mild asthma and isused in approved aerosol antibiotic formulations such as tobramycinsolution for inhalation and aztreonam for inhalation. Eschenbacher W L,Alteration in Osmolarity of Inhaled Aerosols Cause Bronchoconstrictionand Cough, but Absence of a Permeant Anion Causes Cough Alone. Am RevRespir Dis (1984); 129:211-215.

The peak concentrations that one can achieve in the sputum can bepredicted by estimating the mass of drug delivered to the lower airwaysin mg and multiplying by a factor of 30 to get an estimate ofconcentrations. For instance for TOBI, 36 mg is delivered to the lungand the sputum concentrations are approximately 1,000 μg/ml. ForCayston® (Aztreonam Lysine), 30 mg is delivered and the concentrationsare approximately 750 μg/ml. VAP patients have much less sputum averagelikely less than 10 mL, than a patient with CF or bronchiectasis, thataverage over 100 mLs. Therefore, tracheal concentrations are typically10 fold higher in VAP patients compared to sputum concentration in CFand bronchiectasis patients if the same amount of drug is delivered tothe lung.

About 45 mg of amikacin and about 30 mg of fosfomycin are delivered tothe lung if a 6 mL dose (50 mg amikacin and 20 mg fosfomycin insolution) is used in a nebulizer with an expected 15% deliveryefficiency. The predicted concentrations of amikacin would be about13,500 μg/ml, greater than 25 times the MIC 90 for most Gram negativeorganisms. The predicted peak concentrations of fosfomycin would beabout 5400 μg/ml; again more than 25 times greater than the MIC 90 forStaphylococcus aureus based on the similar ratios of deposited drug (inmg) to sputum concentrations in μg/ml of 30 that is seen with tobramycinand aztreonam aerosols after correction for sputum volume differencesbetween CF, bronchiectasis as compared to VAP. With the exception ofpentamidine that has a prolonged half-life in the lung due to binding ofthe drug to surfactant in the alveolar space, the two other FDA-approvedinhaled antibiotics, tobramycin and aztreonam, have an airway half-lifeof about 2 hours. Thus, dosing for aerosolized antibiotics is generallyBID (twice daily) or TID (three times daily) because little therapeuticdrug remains after 5 half-lives or ten hours. Systemic absorption ofdeposited drug is about 10%; thus, even with sputum concentrations onaverage 100-fold greater than what can be achieved with intravenousdrug, the systemic exposure of aerosol antibiotics is on the order of10% of a therapeutic intravenous dose.

Peak concentrations are not a perfect predictor of efficacy. In the caseof amikacin, sputum is known to antagonize the bioavailability ofamikacin and thus doses that achieve at least tenfold the MIC 90-foldhigher are needed for efficacy. Mendelman et al., Aminoglycosidepenetration, in activation, and efficacy in cystic fibrosis sputum. AmRev Respir Dis (1984); 132:761-765). Efficacy is also known to becorrelated with peak concentrations of aminoglycosides, making aerosoldelivery with the high concentrations nearly ideal. In the case offosfomycin, time above the MIC is more important than peakconcentrations. The half-life of inhaled aerosol is on averageapproximately 1.5-2 hours, so a 5400 μg/ml initial dose would be at theMIC 90 of MRSA as early as eight half-lives or about 12 hours.

Twice daily dosing is preferred due to the rapid clearance offosfomycin, and prior study data from aminoglycoside treatment. In aPhase 2 VAP study comparing once-a-day versus twice a dayadministration, aerosolized amikacin as adjunctive therapy to IVantibiotics, twice a day was superior in reducing the need foradditional salvage antibiotics Niederman, et al. NKTR-061 (InhaledAmikacin) Reduces Intravenous Antibiotic Use in Intubated MechanicallyVentilated Patients During Treatment of Gram-Negative Pneumonia. from27th International Symposium on Intensive Care and Emergency MedicineBrussels, Belgium. 27-30 Mar. 2007, Critical Care 2007, 11 (Suppl2):P97). Uniquely challenging bacterial infections subject to treatmentby the compositions of the invention are organisms that are drugresistant, such as MRSA, and those harboring genes that confer bacterialresistance. In particular, genes encoding the carbapenemase enzymes.These beta lactamase enzymes confer resistance to beta lactamantibiotics by hydrolyzation of carbapenems. The New Dehlimetallo-beta-lactamase-1 (NDM-1) is a class B metallo-beta-lactamasethat has spread worldwide and is frequently associated with so-called“super bugs” due to the rapid spread and resistance to antibioticsconferred by the enzyme. Genes encoding NDM-1, or other carbapenemases,can be exchanged between organisms by a variety of methods includingconjugation, plasmid exchange, and bacteriophage transduction. The genescan be incorporated into the bacterial chromosome or borne by a plasmid.Treatment by the methods and compositions of the invention may followidentification of a carbapenemase in a bacterial isolate or by anystandard methodology that establishes infection of a patient with abacteria exhibiting antibiotic resistance. Accordingly, the methods ofthe invention include administering the compositions and utilizing themethods described herein in combination with bacterial diagnostics andin response to identification of a resistant organism in a patient.

An ideal aerosol delivery system for existing mechanical ventilatorswould have the following parameters: the system would be compatible withall ventilator models made of disposable components; capable of creatingsmall particle aerosol size to prevent rain out in the endotrachealtube; and capable of rapid delivery of therapeutic quantities ofantibiotic without creating additional airflow to trigger ventilatoralarm or control systems. A nebulizer with these parameters, theinvestigational PARI eFlow inline nebulizer, yields the data disclosedherein. By vibrating a laser-drilled, thin stainless steel membrane, asmall nearly uniform particle aerosol is created for drug delivery. Thistechnology has been proven in the handheld Altera® device recentlyapproved to deliver aztreonam for inhalation in cystic fibrosis patientswith chronic endobronchial pseudomonas infections. A similar membrane,modified by a smaller hole size and located in a unit that is placedinline with a ventilator inspiratory tubing is preferred. The design isunique with the membrane in the middle of the tubing, with theinspiratory flow freely moving around the membrane to entrain theaerosol as it is created (See FIG. 1). The nebulizer will be runcontinuously, and the estimated lung deposition is 15%. Bias flow, if afeature on the ventilator, will be adjusted to less than 5 liters/minuteto prevent excess flushing of the drug during exhalation.

FIG. 1 is a schematic of a system of the present invention comprised ofa complete airway including a ventilator 1, inspiratory 2 and expiratorylimbs 3, a humidifier 4, an inline nebulizer 5, and a fixture 6 foroperably connecting the system to a patient. The position of thehumidifier 4 is preferably proximate to the inline nebulizer 5, and thenebulizer 5 is most proximate to the patient. The humidifier 4 and thenebulizer 5 are both joined to the airway of the ventilator by a fixturethat is sealed at each point of attachment to the inspiratory limb 2such that additional air is not introduced into the inspiratory limb 2during inspiration by the patient. The antibacterial compositionyielding a hypertonic solution is introduced into the nebulizer 5 foradministration to the patient. Unlike drug administration protocolsprovided in the medical literature, the humidifier 4 is affirmativelyactivated during operation of the nebulizer 5 to achieve the method forreducing the osmolality of the hypertonic solution as described above.As noted above, the humidifier 4 and/or the nebulizer may be activatedby a controller or program operating the ventilator 1, by patientinspiration, or may be continuous during administration of the drugcombination(s).

Because humidification does not need to be turned off during delivery,the small particles grow to an average size of about 3.2 microns afterhumidification, leading to excellent peripheral deposition. Thenebulizer is designed to be in line for the entire treatment course. Theelectronic control unit, the size of a cell phone, is plugged into thewall outlet with a cord that attaches to the nebulizer 5. The nebulizer5 would be inserted near the distal end of the inspiratory tubing towork with any positive-pressure ventilator 1. Unlike a jet aerosoldevice, the system of FIG. 1 would not introduce any additional air andwould avoid hyperinflation or barotrauma in a patient. The disposabledrug/device components eliminate the cost of cleaning, and the one-timeplacement and ventilation of the nebulizer 5 in the system reduces therisk of bacterial contamination of a nebulizer, a known source ofnosocomial infection. Leaving the nebulizer in place between treatmentsminimizes the opening of the ventilator circuit to the environments, awell-known risk for superinfection. In addition, a single patient useprevents any risk of patient-to-patient transmission of resistantbacteria. Drug delivery time would likely be approximately 20 minutes,twice a day.

In the methods of the present invention, a combination of amikacin andfosfomycin is administered from solution at a ratio of amikacin tofosfomycin greater than 1.1, greater than 9:5, greater than 2:1, andpreferably greater than or equal to 2.5-2.6:1. The amikacin isformulated as amikacin chloride as described in Examples 6 and 7 below.The combination of antibiotics is dissolved in a hypertonic solution asdescribed herein and is used to create an aerosol mist having a meanparticle size less than five microns and an osmolality less than 1000mOsm/L. The combination is preferably delivered by placing each in areservoir in the inline nebulizer located within the airway of amechanical ventilator. Alternatively, either component may be deliveredby attaching a drug reservoir such as a dry powder container at a pointwhere inspiration by the patient or movement of air in the ventilatorairway advances drug composition to the patient. The two-partamikacin:fosfomycin formula ideally has a near neutral pH resulting frombalancing the quantity, concentration, pH, and formulation of theindividual components.

Preferably, the nebulizer is sealed in the airway to prevent additionalairflow from being introduced and to permit a combination of the aerosolmist of the antibiotic formulation with humidified air generated by theventilator system. In the system described herein, movement of airthrough the pathway of the ventilator combines humidified air and theaerosol mist containing the antibiotic formulation. Movement of aircontaining the aerosolized drug combination may be triggered by patientinspiration or as part of a continuous or programmed delivery protocolsuch that the nebulizer is in intermittent or continuous operationduring the administration of the antibiotic combination. In each case,the formation of the aerosol is maintained by the apparatus for aduration adequate to deliver bacteriostatic or bactericidal amounts ofthe antibiotic combination to the lung of the patient.

The calculation of the total antibiotic delivery may be achieved by thequantity or concentration of the antibiotic, bactericidal dose, such asthe MIC 90 for any identified organism, or it may be determined throughclinical observation of the organism. As described in connection withFIG. 1 below, the ventilator system typically has an airway that extendsfrom the pressure-generating components of the ventilator through theairway and into the wye fixture that terminates at the patient. Theinline nebulizer may be placed at any point in the airway between thepositive pressure-generating mechanics of the ventilator and thepatient; however, the placement of the nebulizer proximate to thepatient near the ventilator wye piece is preferred. The nebulizer andthe humidification apparatus of the ventilator should be oriented sothat the humidified air causes hygroscopic growth of the individualparticles in the aerosol mist. As noted elsewhere herein, theadvantageous expansion of the aerosol mist particles from an initialsize to an enlarged size, caused by the humidification effect on theradius of each particle, will dictate the location of the nebulizer andthe humidification apparatus. The combination of the humidified air andthe antibiotic solution mist must also achieve reduction in theosmolality as described herein.

In practice, a patient is connected to a ventilator for breathingassistance and the ventilator system is adjusted to provide for acontinuous and controlled airflow based on known physiologicalparameters. The antibiotic composition of the invention is introducedinto a reservoir in the nebulizer and is stored therein until delivery.To administer the antibiotic combination of the present invention, theinline nebulizer is connected to the airway of the ventilator andactivated to create the aerosol mist. Upon delivery, the nebulizergenerates the aerosol mist from a vibrating apparatus disposed therein,typically a vibrating mesh or membrane that has numerous aperturesformed therein to produce particles of a defined size from solution. Thehumidification generator is activated and maintained in operation duringeach delivery of the aerosol mist formed from the hypertonic solutionsuch that the osmolar load is reduced. Thus, the advantage of an inlinenebulizer as described herein is to permit the humidified air in theventilation airway to pass through the nebulizer and to combine with theaerosolized portion of the hypertonic antibiotic combination solution.

Although the embodiment for treatment of VAP and VAT is described hereinin the context of a treatment that occurs while a patient is connectedto a mechanical ventilator system, the compositions of the presentinvention are suitable for administration to a patient who has beenremoved from a mechanical ventilator but continues to suffer a bacterialinfection, typically as a result of the aftermath of a diagnosed VAP orVAT condition. In such cases, the antibiotic composition of the presentinvention can be delivered through an ordinary nebulizer as in the caseof antibiotics delivered to patients suffering from cystic fibrosis. Insuch circumstances, the total composition of the administeredantibiotic, the formulation parameters, and all other characteristics ofa bactericidal treatment regimen are maintained.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of a ventilator and inline nebulizer configured todeliver the compositions and perform the methods of the presentinvention.

FIG. 2 is tracheal aspirates mean (SD) amikacin Cmax.

FIG. 3 is tracheal aspirates mean (SD) fosfomycin.

FIG. 4 is a graph of amikacin concentrations against % inhibitionshowing synergy with fosfomycin.

FIG. 5 is a flow diagram of the manufacturing steps of the amikacinsolution.

FIG. 6 is a flow diagram of the manufacturing steps of the fosfomycinsolution.

DETAILED DESCRIPTION OF THE INVENTION

Amikacin is a preferred aminoglycoside in the ICUs and in ventilatorpatients due to its better activity against Acinetobacter baumanniibacteria than tobramycin. In aerosol studies, a nebulizer dose of 400 mgamikacin led to mean sputum concentrations of 11,900 μg/ml with a widevariability with median <6400 μg/ml. Niederman et al, BAY 41-6551Inhaled Amikacin achieves bactericidal tracheal aspirate concentrationsin mechanically ventilated patients with Gram-negative pneumonia(Intensive Care Medicine 38:263-271, 2012) A Pharmacokinetic Study ATS2010 New Orleans, La.). The vibrating plate nebulizer used in this studytriggered only on inspiration and delivery time of a 2.7 mL formulationaveraged 50 minutes. If run continuously, the PARI vibrating platenebulizer has about 15% delivery efficiency and can deliver a 6 mL dosein 12 minutes. Hahn et al In vitro assessment of a novel nebulizer formechanically ventilated patients based on the eFlow® technology, ISAM2009, Monterey Calif. In the phase 2 CF fosfomycin/tobramycin study, thenebulizer dose of tobramycin was only 20 mg, with estimated 5 mgdelivered to the lung. This illustrates the synergy seen with acombination of fosfomycin and an aminoglycoside. However, relying onsynergy is not appropriate in VAP patients where the bacteria may beresistant to fosfomycin and is a life threatening condition.

The doses of aminoglycosides at first examination may seem excessive;however, sputum macromolecules bind aminoglycosides, so up to 90% ofaminoglycoside is bound and therefore inactive. Therefore, with aerosolaminoglycoside monotherapy, a sputum concentration that is a leasttenfold higher than the MIC 90 is considered necessary, and higher-foldconcentrations, up to 25-fold may provide increased bacterial killing(Mendelman Am Rev Rispir Dis 1985; 132:761-5). Fosfomycin interfereswith the sputum antagonism, (MacCleod, supra), thus even if the bacteriaare fosfomycin-resistant, there may be some clinical benefit to thecombination by increasing the bioactive concentrations of theaminoglycoside.

The optimally effective dose of fosfomycin is likely at least 15 mgdelivered to the lung, with nebulizer doses ranging from 50 to 200 mgdepending on nebulizer efficiency. This is based from the successfulphase 2 CF trial (Trapnell et al., supra), which showed decreasedbacterial density of both pseudomonas and Staphylococcus aureus in thesubset of patients who were co-infected, with approximately 20 mgdelivered to the lung. In this trial, an estimated 40 mg delivered doseof fosfomycin was more efficacious in killing staphylococcus than theestimated 20 mg dose, showing that a higher dose may be better. The mostsoluble fosfomycin salt is the disodium salt and is preferable althoughother salts are possible—such as calcium and tromethamine.

The recent development of vibrating plate nebulizers, particularly oneby PARI, enables particle sizes less than 5 microns. See WO2005/048982A2. Membranes having a plurality of small apertures thereincan produce mean particle sizes less than 5 microns and in the range of3.5 microns. This is accomplished by making the porous holes smallerduring the laser drilling process. Other vibrating plate membranes byPARI have a 4.5 micron average size particle as does the vibrating platenebulizer introduced by Aerogen/Nektar. Similarly, there are smallparticle jet nebulizers that can produce 2 3 micron size particles.Current ultrasonic nebulizers produce an average particle size of 5microns using a 2.7 Mhz driving frequency. Ultrasonic nebulizers cancreate smaller particles by increasing the frequency of the ultrasonicgenerator. No high frequency (2.3 Mhz) nebulizers are currentlycommercially available in the United States or Europe at this time, butwould have a 2-3 micron particle size. In addition, ultrasonicnebulizers heat the nebulizer solution and this may lead to drugdegradation during therapy, for this reason, their use has fallen out offavor.

The present invention includes the use of humidification as a techniqueto improve the tolerability of hypertonic solutions delivered as anaerosol. The creation of an aerosol with a small particle size from ahypertonic solution can produce a composition of small particles thatcarry a desirable therapeutic dose but are poorly tolerated due to ahigh osmolality, i.e., on the order of threefold or greater of normalosmolality, (e.g., ≧930 mOsm/kg). Adding humidification to the aerosolyields an aerosol composition that has a reduced osmolality and ispreferably close to isotonic or less than twofold normal osmolality(e.g., <620 mOsm/kg). The humidification is created by an inlinehumidifier to decrease the osmolality to a range from greater thanthreefold to less than twofold normal osmolality and may vary dependingon the nature of the original hypertonic solution. The particle size ofnon-humidified aerosol such as the hygroscopic growth of a 4 micronparticle may lead to much more dilution than growth of sub 3 micronparticle. In such hypertonic solutions, the permanent ion in solution ispreferably greater than 40 mequil/L. The humidification can be appliedto aerosols formed from a variety of hypertonic solutions where pairedtolerability is desired. Examples include any small molecular weightdrugs that require high concentrations for efficacy, or compounds thatare salts with multiple anions or cations that create a high osmolarload in solution.

In the aspect of the invention below, aminoglycoside/fosfomycincombinations are hypertonic on administration but close to isotonic upondelivery by the advantage of increased humidification compared toambient air. For instance, if the particle size grows on average from3.5 to 4.5 microns, the dilution is a function of the cube of the radiusor 4.91/11.3. Therefore, the use of small particle aerosol withsubsequent hygroscopic growth due to humidification would substantiallyreduce the osmotic load on the lung. With a larger initial particlesize, the effect would be similar. For example, the growth from a 5 to 6micron particle would lead to a dilution of 15.6/27. If particles areallowed to grow much larger than 5 microns, tolerability is not theprimary issue, as little will be deposited in the airways due to “rainout” in the ventilator and endotracheal tubing. This was shown in theseminal studies by Palmer (supra) on the deleterious effect ofhumidification on total drug delivery. These studies mostly utilized jetnebulizers that have an average of 4-5 micron particles prior to growthdue to humidification. The hygroscopic growth was responsible for rainout and less drug delivered to the airways. For instance, applying theratio of 4.91/11.3, if a hypertonic solution is used with a nebulizerthat has a 3.5 micron average particle, an osmolality of up to 710 wouldbecome, on average, isotonic. Slightly hypertonic formulations can betolerated by the lung, and it is likely a formulation with an osmolalityof up to 800 would be well tolerated by applying the humidificationtechnique described herein.

The PARI inline nebulizer designed for ventilator use can be outfittedwith a small pore membrane, has a current volume capacity of 10 mL, andhas a rate of delivery of 0.5-0.6 ml/minute. Although it is currentlynot configured for triggering on inspiration, a nebulizer may be soconfigured when operably connected to the control system of theventilator. Particle size would be estimated at 3.2 microns. Aformulation of 10 mL, with 100 to 300 mg fosfomycin and 300 to 600 mg ofamikacin at the 15% efficiency rate would provide adequate killing forStaphylococcus aureus and Pseudomonas. An ideal formulation wouldcontain at least 20 meq/I of chloride anion after dilution. Theestimated osmolality of a solution of 50 mg/mL amikacin and 20 mg/mL offosfomycin, with chloride anion, adjusted to a pH between 4.5 and 7.5 isapproximately 750-850 osm/L. If diluted by humidification, this wouldlikely be close to the isotonic range when deposited in the airways. Tovary the delivered dose, a smaller or larger volume could be used, oralternatively or in combination, trigger delivery on inspiration phaseof breathing to increase the deposition amount.

Example #1 Preparation of Fosfomycin/Amikacin Solution forAerosolization

A solution having a ratio of amikacin to fosfomycin of equal to orgreater than 2.5-2.6:1 is prepared as follows: Fosfomycin disodium(12.90 g, 10.00 g free acid) was dissolved in 250 mL of water and the pHwas adjusted to 7.41 by the dropwise addition of 4.5 N HCl (estimated 1mL). 25 gm Amikacin base was added to the resulting solution. The pH ofthe solution was adjusted to 7.60 by the addition of 4.5 N HCl (totalamount of 4.5 N HCl was 1.7 mL). The solution was diluted to 500 mL withwater and filtered through a 0.2 μm Nalge Nunc 167-0020 membrane filterfor sterility. The chloride content can be calculated by using 1.7 mL of4.5N HCl in 50 L total for a total 306 mg chloride. As 1 mEq CI=35.5 mgin I L then in 50 mL 1 mEq CI=1.775 mg. Therefore, 306 mg/1.775 mg=172.4mEq/L. The osmolality of this formulation was measured at 592 mOsm/kg,which is above the normal physiologic value of 310 mOsm.

Example #2 Reduction of the Osmolality of the Solution by Humidification

A solution having a fosfomycin/amikacin ratio of 2.5-2.6-1 amikacin tofosfomycin was prepared as above. Using an inline electronic vibratinglate nebulizer (PARI, Starnberg GR), the formulation was nebulized indry (4%) and humid (100%) humidity. The mean particle size, as measuredby Malvern X laser particle sizer, was 2.9 μm under dry conditions,increasing to 3.2 μm under 100% humidity.

Since the volume of sphere is function of the third power of the radius,the following equation yields the dilution factor:

$\frac{1.45 \times 1.45 \times 1.45}{1.6 \times 1.6 \times 1.6} = 0.75$

Thus, the formulation on average is diluted by a factor of 0.75,indicating the delivered formulation has an osmolality of 592×0.75=444mOsm/Kg.

Example #3 Randomized, Double-Blind, Placebo-Controlled, Dose-EscalationPhase 1 b Study of Aerosolized Amikacin and Fosfomycin Delivered Via thePARI Investigational eFlow® Inline Nebulizer System in MechanicallyVentilated Patients

A dry powder fosfomycin, liquid amikacin solution can be prepared by useof 200 mg neat dry powder disodium fosfomycin filled in a glass vial ortwo-part dry liquid syringe. In either a separate syringe, blow fillseal container, or a two-part syringe, 500 mg of amikacin base dissolvedin 10 mL of sterile water, with the pH adjusted to a range of 4.5 to 7.5with HCl. The two components are then mixed together giving a solutionwith 20 mg/mL fosfomycin, 50 mg/ML amikacin. The osmolality of thesolution would be approximately 600 mOsm/Kg, but could vary up to 10%depending on the amount of HCl used to adjust the pH of the amikacinsolution. Also by employing the chlorine counter anion with the amikacinbase, the sulfate salt of amikacin is not used.

A treatment regimen was designed to control safety, efficiency,tolerability and to further elucidate systemic and tracheal aspiratepharmacokinetics of nebulized amikacin/fosfomycin in patients with aclinical diagnosis of VAP following delivery of 2 mL, 4 mL, 6 mL, 8 mL,and 10 mL and doses via the PARI Investigational eFlow® Inline NebulizerSystem in mechanically ventilated patients.

Adult patients having a clinical diagnosis of VAP, with a Gram-positiveor Gram-negative organism in a tracheal aspirate sample were expected tobe on mechanical ventilation for ≧3 days.

Each adult patient received 3 escalating doses of a mixture of 50 mg/mLamikacin and 20 mg/mL fosfomycin, with doses separated by 24±2 hours(Table 1). On Day 3, patients received 2 blinded, randomized treatments(amikacin/fosfomycin and volume-matched placebo [0.9% normal saline]),separated by 2 hours.

All treatments were administered with a single patient, multi-treatmentnebulizer (Investigational eFlow® Inline Nebulizer System; PARI PharmaGMBH, Starnberg, Germany), positioned in the inspiratory tubing betweenthe ventilator and the patient. The nebulizer remained inline until alltreatments were delivered. As documented herein, the nebulizer has avibrating perforated membrane and generates an aerosol with smalldroplets and narrow size distribution, which is optimal for depositionin the lower airways.

Concentrations of amikacin and fosfomycin were measured in trachealaspirate and plasma samples obtained during the 24 hours after eachdose, and the maximum concentration (C_(max)) was determined. Adverseevents were assessed from the first dose until 24 hours after the lastdose was received.

TABLE 1 Study Dosing Schedule Cohort # Dose of Amikacin (50mg/mL)/Fosfomycin (20 mg/mL) (Patient #) 2 mL 4 mL 6 mL 8 mL 10 mL 12 mL1 (1-3) X X  X^(a) 2 (4-6) X X  X^(a) 3 (7-9) X X X^(a) Each patientreceived 3 treatments of amikacin/fosfomycin with treatments separatedby 24 ± 2 hours. ^(a)On Day 3, patients received blinded, randomizedtreatment with amikacin/fosfomycin and with volume-matched placebo, withthe 2 treatments separated by 2 hours.

TABLE 2 Bacteria Cultured from Tracheal Aspirates Number of Patientswith Isolates^(a) Bacterial Isolates (N = 7) Staphylococcus aureus 3Citrobacter koseri 1 Enterobacter cloacae 1 Escherichia coli 1Haemophilus influenzae 1 Proteus mirabilis 1 Serratia marcescens 1^(a)Each patient had 1-2 different bacteria cultured from their trachealaspirate.

TABLE 3 Concomitant Treatment with Intravenous Antibiotics Number ofPatients Treated^(a) Antibiotic Treatment (N = 7) Ciprofloxacin 6Piperacillin/tazobactam 6 Vancomycin 4 Cefepime 1 Cefazolin 2Ceftriaxone 2 Erythromycin 2 Gentamicin 2 Flucloxacillin 1 Meropenem 1Moxifloxacin 1 ^(a)Each patient received concomitant treatment with 3-5intravenous antibiotics.

In tracheal aspirate samples obtained from each patient after each doseof amikacin/fosfomycin solution for nebulization. Each amikacin Cmaxvalue was 89 fold higher than the minimum inhibitory concentration for90% (MIC₉₀; 32 μg/ml) of 1477 Pseudomonas aeruginosa isolates describedin a recently published isolate collection. Zhanel et al., DiagnMicrobiol Infect Dis 2011, 69:291.

Mean amikacin Cmax after the 6 mL dose was 406-fold higher than the MIC90 for P. aeruginosa (FIG. 2). Each fosfomycin Cmax value was ≧54-foldhigher than the published MIC 90 value for 148 isolates ofmethicillin-resistant Staphylococcus aureus (MRSA; 32 μg/ml). Meanfosfomycin Cmax after the 6 mL dose was 281-fold higher than the MIC 90for MRSA (FIG. 3). Six hours after dosing, all fosfomycin trachealaspirate concentrations remained ≧2.2-fold above the MIC 90 for MRSA.Plasma concentrations were >2000-fold lower than concentrations intracheal aspirates: The highest observed amikacin plasma concentrationwas 1.4 μg/mL; this is less than the recommended peak concentration (35μg/mL) after a systemic dose. The highest observed fosfomycin plasmaconcentration was 0.8 μg/ml. No adverse event was considered by theclinical investigator related to study-drug treatment. No changes inoxygen saturation or peak airways pressures were noted in response tostudy drug. All patients were alive 28 days after completing the study.High tracheal aspirate concentrations of amikacin and fosfomycin wereachieved in mechanically ventilated patients with VAP after aerosolizedadministration of amikacin/fosfomycin solution for nebulization with aninline nebulizer system. Systemic exposures to amikacin and fosfomycinwere much lower than tracheal aspirate levels. Amikacin/fosfomycinsolution for nebulization was well tolerated.

The combination antibiotic amikacin/fosfomycin (50 mg/mL amikacin and 20mg/mL fosfomycin) formulation was delivered via the PARI InvestigationaleFlow® Inline Nebulizer System in mechanically ventilated patients. Aplacebo: 0.9% normal saline, having a volume matched to the antibioticdosing schedule was delivered via the PARI Investigational eFlow® InlineNebulizer System in mechanically ventilated patients.

The eFlow® Inline Nebulizer System was positioned in the inspiratorytubing between the Puritan Bennett 840 Ventilator and the patient. Oncein place, the nebulizer remained inline until all study-drug doses weredelivered. Humidification continued during the nebulization of theformulation and the delivery of the entire dose.

Patients are male or female between 18 years and 80 years of age withclinical diagnosis of VAP or VAT, a Gram-positive or Gram-negativebacteria on Gram stain of the tracheal aspirate and were expected to beon mechanical ventilation for at least three days.

These results support further clinical trials of amikacin/fosfomycinsolution for nebulization in mechanically ventilated patients with VAPor VAT.

These results demonstrate that high sputum concentrations of amikacinand fosfomycin were achieved in mechanically ventilated patients withVAT or VAP after aerosolized administration with an inline nebulizersystem.

Example #4 Clinical Study for VAT/VAP

A GLP (Good Laboratory Practice) study was performed using 24 beagledogs allocated to four dose groups (three males and three females pergroup) and exposed to aerosol generated with the PARI InvestigationaleFlow® Inline Nebulizer System using a closed-faced mask fitted with amouth tube. The aerosols contained either control (water for injection)in Group 1 or a combined formulation containing 50 mg/mL amikacin and 20mg/mL fosfomycin pH adjusted with HCl for Groups 2 to 4. Aerosolconcentrations were determined on Days 1 and 7. The treatment period wasfor seven days with termination of the dogs on Day 8. The average dailyachieved dose of amikacin/fosfomycin for each group was 32.1:12.4mg/kg/day (a 2.59:1 ratio) (Group 2); 63.0:24.7 mg/kg/day (92.55:1ratio) (Group 3); and 116.8:47.5 mg/kg/day (92.46:1 ratio) (Group 4).The highest estimated pulmonary dose was 29.2 mg/kg/day amikacin and11.9 mg/kg/day fosfomycin. The particle size distribution (MMAD [MassMedian Diameter]) based on analytical methods was determined to berespirable averaging 2.80 μm (GSD=1.778) for amikacin and 2.75 μm(GSD=1.670) for fosfomycin.

The aerosol was well tolerated by all dogs. There were notreatment-related adverse effects based on clinical observations, bodyweights, food consumption, ophthalmoscopy, or electrocardiography. Anychanges to clinical pathology values observed were attributed to normalanimal variation. No treatment-related abnormalities were observed onnecropsy. No treatment-related adverse findings were observed uponhistologic evaluation of tissues.

Toxicokinetic parameters were estimated using WinNonlin pharmacokineticsoftware version 5.2.1 (Pharsight Corp.). A non-compartmental approachconsistent with the extravascular route of administration was used forparameter estimation. All parameters were generated from individualamikacin and fosfomycin concentrations in plasma from Days 1 and 7.Plasma amikacin and fosfomycin concentration vs. time profiles wereconsistent with the inhalation dose route whereby a post-dose absorptionphase was followed by a biphasic bi-phasic decline in plasmaconcentrations. Systemic exposure to both amikacin and fosfomycin wasgenerally comparable between males and females and there was no clearindication of accumulation following repeat dosing. The peak plasmalevels (Cmax) for the high dose level on Day 7 ranged from 13.2 to 39.3μg/ml for amikacin and 8.7 to 28.73 μg/ml for fosfomycin.

Based on the results of the study, significant exposure occurredfollowing aerosol exposure to beagle dogs with no adverse effectsobserved over the 7-day treatment period. The NOAEL was considered to be116.8 amikacin and 47.5 fosfomycin mg/kg/day delivered as a combinationantibiotic aerosol. This is approximately 30-fold the estimated exposureto humans.

Example #5 Synergism of the Amikacin and Fosfomycin Combination AgainstResistant, Gram-Negative Pathogens

Sixty-two amikacin-resistant strains were selected from a worldwideantimicrobial surveillance collection (SENTRY), which contains 35,000organisms from six continents (56/62 organisms collected in 2011). The62 isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, andKlebsiella pneumoniae had an amikacin minimum inhibitory concentration(MIC) of >32 μg/ml μg/mL (Clinical and Laboratory Standards Institute[CLSI]: intermediate or resistant; European Committee on AntimicrobialSusceptibility Testing [EUCAST]: resistant). Each isolate was testedagainst amikacin (0.25-1024 μg/ml), fosfomycin (0.1-409.6 μg/ml), andamikacin/fosfomycin (5:2 ratio) using CLSI methods (agar dilution withsupplements). Control strains included a range of MIC values (amikacin:0.25-1024 μg/ml; fosfomycin: 0.1-409.6 μg/ml).

For 21 A. baumannii, 21 P. aeruginosa, and 20 K. pneumoniae strains,amikacin (Table 1) and fosfomycin (Table 2) MIC values were reduced withthe amikacin/fosfomycin combination. For control stains, 100% ofamikacin and 91.7% of fosfomycin MIC values were within publishedranges.

For A. baumannii strains, the effect was most pronounced for strainswith high amikacin resistances (MIC, >1024 μg/ml); for these 5 isolates,amikacin MIC values were reduced to <256 μg/ml with theamikacin/fosfomycin combination. For 11 of 21 P. aeruginosa strains,amikacin MIC values remained stable (±one log 2 dilution step) with theaddition of fosfomycin. For the other 10 strains, amikacin MIC valuesdecreased >fourfold when fosfomycin was added. For 9 of 20 K. pneumoniaestrains, amikacin MIC values remained stable with the addition offosfomycin. For the other 11 strains, amikacin MIC values decreased morethan fourfold when fosfomycin was added (decrease >32-fold for 6/11strains).

Overall, median MIC values for amikacin (Table 1) and fosfomycin (Table2) each decreased twofold with the amikacin/fosfomycin combination.Addition of fosfomycin reduced the amikacin concentration required toinhibit all 62 isolates from >1024 to <256 μg/mL (FIG. 4).

Combining amikacin in a 5:2 ratio with fosfomycin significantly enhancedthe potency of amikacin against 62 Gram-negative, amikacin-resistantpathogens. Interactions between amikacin and fosfomycin varied byisolate, and ranged from non-detectable to high-level synergy. Theseresults support development of the amikacin/fosfomycin combination foraerosolized administration where high drug levels can be achieved.

TABLE 1 Amikacin MIC values (±fosfomycin) Mediana (range) Amikacin MIC,μg/mL Median (range) Amikacin MIC, μg/mL Amikacin/ No. Isolates AmikacinFosfomycin (5:2) A. baumannii 21  8 (32, >1024)  8 (32, 256) P.aeruginosa 21 128 (32, >1024) 64 (8, 256) K. pneumoniae 20 256(32, >1024)  32 (16, 128) All 62 128 (32, >1024) 64 (8, 256) a. Median =MIC50; Mediana (range) Amikacin MIC; μg/mL; Median′ (range) AmikacinMIC, μg/ml

TABLE 2 Fosfomycin MIC values (±amikacin) Median^(a) (range) FosfomycinMIC; μg/mL No. Amikacin/ Isolates Fosfomycin Fosfomycin (5:2) A.baumannii 21  204.8 (204.8, 204.8)  25.6 (12.8, 102.4) P. aeruginosa 2151.2 (3.2, 102.4) 25.6 (3.2, 102.4) K. pneumoniae 20  25.6 (12.8, 204.8)12.8 (6.4, 51.2)  All 62 51.2 (3.2, 204.8) 25.6 (3.2, 102.4) ^(a)Median= MIC50; Mediana (range) Fosfomycin MIC, μg/ml

Example 6 Amikacin Chloride Formulation from Amikacin-Base and pHBalance

Inhalation solutions are designed to be tolerable when administered, andthere is substantial literature identifying a few critical parametersthat influence tolerability. The pH of the solution, the osmolality ofthe solution, and the presence of permeable anions in the solution alldetermine whether a solution can be acceptably delivered to the lungswithout triggering adverse reactions (cough, bronchospasms, etc).

A two-part formulation of amikacin and fosfomycin (which is mixedimmediately prior to use) was designed so that the final mixed solutionwould be as close to neutral in pH as possible, while maximizing thestability of each component. Fosfomycin is most stable under basicconditions, which requires the amikacin solution to be lower in pH, sothat the resulting mixture is neutral.

An acidic, citrate-buffered formulation of amikacin (based on thecommercial injectable formulation) was compared to an unbufferedformulation at pH 7. A mixture experiment was performed by mixing eachinto fosfomycin solutions at increasingly basic pH values (which enhancethe solutions stability of fosfomycin). Results are in Table 3.

TABLE 3 Final solution pH after mixing of 40 mg/mL Fosfomycin with 100mg/mL Amikacin Fosfomycin Fosfomycin Fosfomycin pH 8 pH 9 pH 10 Amkacin,100 mg/mL, 7.51 7.55 7.56 pH 7 (WFI) Amikacin, 100 mg/mL, 6.82 6.87 6.86pH 5.5 (citrate)

Another formulation uses fosfomycin disodium as a powder mixed with theamikacin solutions. The two amikacin formulations were mixed with theappropriate amount of fosfomycin disodium powder and the resultingsolution pH was evaluated. Results are in Table 4.

TABLE 4 Final solution pH after mixing of 200 mg of Fosfomycin with 8 mLof Amikacin at 50 mg/mL ~210 mg Fosfomycin disodium powder Amkacin, 50mg/mL, 7.58 pH 7 (WFI) Amikacin, 50 mg/mL, 6.87 pH 5.5 (citrate)

The data show that all final solutions fall within a pH range consideredtolerable with the lung physiology (˜4.2-8.0). Amikacin at pH 7 whenmixed with fosfomycin at pH 8, 9, or 10, produces a final solution pH of7.51-7.56. Fosfomycin is increasingly stable as the pH becomes morebasic. Therefore, more basic formulations of fosfomycin can be createdif needed.

The data also support a powder formulation comprising amikacin solutionsmixed with the required amount of fosfomycin disodium powder to yield aresulting solution with an acceptable pH (6.87-7.58).

A two-part liquid formulation with the amikacin component at a neutralpH is also beneficial and would be well tolerated by a patient.

Example 7 Manufacturing Plan for Amikacin/Fosfomycin Combination

The Amikacin Fosfomycin Inhalation System (AFIS) consists of amikacinsolution (AMS) (3 mL sterile unit dose ampoule, 100 mg/mL), fosfomycinsolution (FFS) (3 mL sterile unit dose ampoule, 40 mg/mL) and the PARIInvestigational eFlow® Inline Nebulizer System. AMS and FFS weredispensed to the nebulizer reservoir to create the Amikacin FosfomycinAdmixture (AFA). The nebulizer delivered the aerosolized AFA directlyinto the inspiratory arm of a ventilator for patients receivingmechanical ventilation.

Amikacin Base, EP is compendial (Ph Eur) and is manufactured by ACSDobfar S.p.a under DMF 13762. The API is manufactured in Bergamo, Italy.Fosfomycin Disodium is compendial (Ph Eur) and is manufactured byErcros, S.A. under DMF 14341. The API is manufactured in Madrid, Spain.Rather than commercial amikacin sulfate, the amikacin base is thestarting material. Amikacin base has a pH of approximately 11, and largeamounts of HCl are required to neutralize the pH. In the two-partformulation (used to combine with equal volumes of 40 mg/mL fosfomycinto generate the final formulation of 50 mg/mL amikacin, 20 mg/mLfosfomycin), the amikacin 100 mg/mL solution has a final pH of 7.5-7.6and has an osmolality of approximately 533 mOsmol/kg, of which onlyapproximately 170 mOsmol/kg is due to the amikacin, the balance is fromthe chloride anion. This concentration of chloride anion provides thepermeant anion that will prevent cough.

In contrast, the starting pH of amikacin sulfate is 3.5-5.5 and littleor no HCl is needed to neutralize the pH of the solution. Note that inthe Neiderman paper (supra) amikacin sulfate was delivered as an aerosolto patients with VAP, and bronchospasm was reported in some patients. Incontrast, none was seen in the study presented in Example 3. Asdescribed above, a final formulation has an amikacin concentration of 50mg/ml and chloride anion concentration of approximately 265 meq/liter.The low end of an acceptable range would be an amikacin concentration of25 mg/ml and chloride anion concentration of approximately 130meq/liter; however, as low as 30 meq/liter could be used with anotheranion in addition to chloride. The high end of the amikacinconcentration would be 100 mg/ml and a chloride anion concentration ofapproximately 540 meq/liter. This formulation yields an osmolality of900 mOsmol/L, and any increase would prevent dilution of the formulationwith humidity sufficient to make it tolerable.

The flow diagram depicting the steps of the manufacturing process ofamikacin solution is provided in FIG. 5. The flow diagram indicateswhere each raw material enters the manufacturing process.

The amikacin solution is manufactured as follows:

-   -   1. Add the calculated amount of Water for Injection (WFI) to the        stainless steel tank.    -   2. Add the calculated amount of amikacin to the tank and mix        until dissolved.    -   3. Add 90% of the calculated amount of HCl and mix for 5-10        minutes.    -   4. Titrate the amikacin-HCl solution with remaining HCl until        solution reaches pH 7.0±0.3.    -   5. Bring the solution to final mass with WFI and mix for 10-15        minutes.    -   6. Filter the solution through a 0.22 micron filter into a        holding tank.    -   7. Filter the solution through two 0.22 micro filters in series.    -   8. Use Blow/Fill/Seal equipment to form the LDPE ampoule, fill        to a target weight and then seal the ampoule.    -   9. Perform 100% visual inspection and integrity testing on LDPE        ampoule.    -   10. Foil overwrap ampoules individually.    -   11. Perform 100% leak detection on foil overwrapped ampoules.

The flow diagram depicting the steps of the manufacturing process offosfomycin solution is provided in FIG. 6. The flow diagram indicateswhere each raw material enters the manufacturing process.

The fosfomycin solution is manufactured as follows:

-   -   1. Add the calculated amount of WFI to the stainless steel tank.    -   2. Add the calculated amount of fosfomycin to the tank and mix        until dissolved.    -   3. Add 90% of the calculated amount of HCl and mix for 5-10        minutes.    -   4. Titrate the solution with remaining HCl until solution        reaches pH 8.0±0.3.    -   5. Bring the solution to final mass with WFI and mix for 10-15        minutes.    -   6. Filter the solution through a 0.22 micron filter into a        holding tank.    -   7. Filter the solution through two 0.22 micro filters in series.    -   8. Use Blow/Fill/Seal equipment to form the LDPE ampoule; fill        to a target weight; and then seal the ampoule.    -   9. Perform 100% visual inspection and integrity testing on LDPE        ampoule.    -   10. Foil overwrap ampoules individually.    -   11. Perform 100% leak detection on foil overwrapped ampoules.

Option 1—Use Phase 2 Manufacturing Process

In a scaled-up commercial the manufacturing process may be transferredto other contract manufacturing sites that specialize in Blow/Fill/Sealtechnology.

Such that both solutions will be contained in one overwrapped ampoulehaving dual chambers and a single opening. In use, both solutions willbe dispensed into the nebulizer at the same time.

All references cited herein are specifically incorporated by reference.

What is claimed is:
 1. A method to administer a combination ofantibiotics during mechanical ventilation of a patient to prevent oralleviate ventilator associated pneumonia (VAP) or ventilator associatedtracheal bronchitis (VAT) comprising: establishing a positive pressurein the airway of a mechanical ventilator to provide assisted breathingto the patient, wherein the mechanical ventilator has means forintroducing humidified air to the airway, connecting an inline nebulizerhaving an outlet into the airway, activating the nebulizer such that thenebulizer introduces an aerosol mist in the airway, wherein the mist iscreated from a solution stored in a reservoir therein and wherein theaerosol mist formed in the nebulizer is comprised of a distribution ofparticles having a mean particle size less than 5 microns; wherein thesolution is a combination of a bactericidal combination of amikacin andfosfomycin at a ratio of amikacin to fosfomycin greater than 1.0:1.0 andin a hypertonic solution having at least 30 mEq/L permeant ion and anosmolality less than 1000 mOsmoi/L; combining the aerosol mist with thehumidified air such that the mean particle size of the aerosol mistincreases by at least 10% and the osmolality decreases by at least 25%;and maintaining activation of the nebulizer until a dosage of amikacinand fosfomycin is delivered to achieve a MIC 90 for an organism selectedfrom a Gram-positive organism and a Gram-negative organism andcombinations thereof.
 2. The method of claim 1, wherein the step ofactivating the inline nebulizer provides continuous creation of theaerosol mist until the reservoir is substantially emptied.
 3. The methodof claim 1, wherein the step of connecting the in line nebulizer createsa sealed airway such that additional air is not introduced to the airwayduring activation of the in-line nebulizer.
 4. The method of claim 1,wherein the step of combining the aerosol mist with the humidified airoccurs substantially within a housing of the nebulizer, or in a spaceimmediately adjacent thereto, and within the airway.
 5. The method ofclaim 1, wherein humidified air is introduced into the airway duringsubstantially the entire duration of the step of activating thenebulizer.
 6. The method of claim 2, wherein the total dosage ofamikacin in solution converted to the aerosol mist is between 150 and600 mg.
 7. The method at claim 2, wherein the total dosage of fosfomycinin solution converted to the aerosol mist is between 100 and 300 mg. 8.The method of claim 1, wherein the Gram-positive organism is MRSA. 9.The method of claim 1, wherein the Gram-negative organism harbors aplasmid expressing a carbapenemase.
 10. The method of claim 9, whereinthe carbapenemase is NDM-1.